This collaborative, interdisciplinary project renewal is focused on the role of the M protein in the virulence of group A Streptococcus (GAS, S. pyogenes). The Gram-positive bacterial pathogen GAS is a leading cause of global morbidity and mortality. With an estimated >500,000 annual deaths, GAS ranks among the top 10 causes of mortality from infectious disease. The M protein is the major surface-associated virulence factor of GAS and has a significant role in the entire range of conditions caused by GAS. The M protein is also a major target of neutralizing immunity, but because of its antigenic variability (>220 distinct M types), immunity to GAS is typically narrow, being limited to the M type of the infecting strain. The overarching theme of this renewal proposal is the interplay between functional indispensability and antigenic variation. Functionally essential regions of microbial virulence factors are excellent targets for intervention, because these regions tend to be strictly conserved and unable to escape intervention without surrendering function (i.e., Achilles' heels of the protein). But due to selective immune pressure, these Achilles' heels are often hidden by antigenically variable regions. The GAS M protein presents an exemplar of this evolutionary conflict between functional indispensability and sequence variability. We have shown recently that sequence conservation lies hidden within the antigenic variability of the M protein, and that the key to detecting hidden conservation is through three-dimensional structural studies. This revised renewal proposal seeks to identify several potential Achilles' heels in M proteins that can serve as targets for therapeutic intervention or vaccine design (no GAS vaccine exists at present). In our renewal application, we will leverage our published and preliminary results to examine functionally important interactions of M proteins with human C4b-binding protein (Aim 1), the antimicrobial cathelicidin LL-37 (Aim 2), and fibrinogen (Aim 3). This project sustains a uniquely synergistic and long-standing collaboration between the complementary efforts of the Ghosh (biochemistry and structural biology) and Nizet (bacterial pathogenesis and innate immunity) laboratories to study GAS virulence from atoms to animals. Once completed, these studies will provide essential knowledge applicable to the design of novel therapeutics or preventive strategies against GAS.